Parkinson’s disease shows a heterogeneous course and different clinical subtyping systems have been described. To compare the capabilities of two clinical classification systems, motor-phenotypes, and a simplified clinical motor-nonmotor subtyping system, a cohort was included at mean 7.9 ± 5.3 years of disease duration, classified using both clinical systems, and reexamined and reclassified at the end of an observation period. Time-points were retrospectively extracted for five major disease milestones: death, dementia, Hoehn and Yahr stage 5, nursing home living, and walking aid use. Eighty-nine patients were observed for 8.1 ± 2.7 years after inclusion. Dementia developed in 32.9% of the patients and 36.0–67.4% reached the other milestones. Motor-phenotypes were unable to stratify risks during this period, but the worst compared with the more favorable groups in the motor-nonmotor system conveyed hazard ratios between 2.6 and 63.6 for all milestones. A clear separation of risks for dying, living at the nursing home, and reaching motor end-stage was also shown when using only postural instability and gait disorder symptoms, without weighing them against the severity of the tremor. At reexamination, 29.4% and 64.7% of patients had changed classification groups in the motor-phenotype and motor-nonmotor systems, respectively. The motor-nonmotor system thus stratified risks of reaching crucial outcomes in mid–late Parkinson’s disease far better than the well-studied motor-phenotypes. Removing the tremor aspect of motor-phenotypes clearly improved this system, however. Classifications in both systems became unstable over time. The simplification of the motor-nonmotor system was easily applicable and showed potential as a prognostic marker during a large part of Parkinson’s disease. Time and pattern of progression to such PD end-stages is heterogeneous2 and various classification systems have been developed, trying to elucidate these differences11.In later stages of Parkinson’s disease (PD), some patients develop dementia or permanently need to use a wheelchair , whereas others never become as severely affected4. However, nonmotor symptoms are found to hold prognostic information15 that are crucial for accurate PD prognostication16 and have been successfully implemented in several PD classifications9. Unfortunately, the data-driven approaches commonly used can make replication difficult17, and identifying scales and measures that are easily applied in a normal out-patient appointment, and also hold high reproducibility between examiners and patients from different ethnicities, can be challenging18. Recently, a study constructed an algorithm for a new clinical subtype classification system, based on cluster analysis of both motor and nonmotor findings3. Patients of the different groups were shown to vary in cerebrospinal fluid biomarkers3 and clinically and radiologically evaluated disease progression21. Improved clinical feasibility and confirmation of this system with population-based inclusion and longer time of observation have been requested22. The prognostic capabilities of these subtypes have been assessed in two other longitudinal PD cohorts, grouping patients close to diagnosis21, but, to the best of our knowledge, the long-term efficacy of this system when applied in mid-stage PD has not yet been examined. In this study, we adapted the motor-nonmotor subtyping system to facilitate clinical use and applied both this system and motor-phenotypes in patients with mid and late stages of PD from a cohort with long follow-up, comparing risks for reaching relevant PD milestones.One frequently used classification system, motor-phenotypes, is based on the predominant motor findings in PDBaseline examination was performed at 7.9 ± 5.3 (mean ± SD) years after disease onset in 89 patients with HY-stage 2.8 ± 1.1. Follow-up data covering the following 8.1 ± 2.7 years was extracted from medical records, more specifically until 16.0 ± 5.4 total years of disease duration and 75.7 ± 8.0 years of age. In the most benign groups in each system (mild-motor-predominant and tremor-dominant), there were fewer patients that reached the assessed milestones, but they also had younger-onset ages, a higher proportion of women, and shorter disease duration . The risk for developing dementia was seven times higher among patients with hallucinations at baseline compared to those without  after 12.6 ± 5.9 years of disease . When both classification systems were re-applied, 29.4% and 64.7% of the re-examined patients changed classification groups in the motor-phenotype and motor-nonmotor systems, respectively  III score were lower in patients with re-examination compared to those without: 55.3 ± 8.2 vs 62.4 ± 8.8 years, and 15.3 ± 7.3 vs 26.2 ± 11.4 points, respectively. Nonmotor symptoms questionnaire (NMSQ) scores showed a slight difference between re-examined and not re-examined patients, 9.0 ± 4.2 vs 10.4 ± 5.2 points, respectively. Nine women and 27 men had died before re-examination and 53.0% of reexamined patients were men compared to 60.1% at baseline. Hallucinations at baseline were correlated with individual Addenbrooke’s Cognitive Examination Revised (ACER) scores at reexamination  had then developed dementia. Previous studies on patients with 20 years of PD duration have reported high diversity regarding cognitive outcome; 83% of PD patients developed dementia in one longitudinal study30. Lack of usefulness of the motor-phenotypes could be due to a confounding effect of disease stage31. A large proportion, 66.3%, of the present cohort had reached PIGD motor-phenotype at baseline, which likely diminishes the usefulness of this system when it is applied in the middle stage of PD, as in the present study. This is not surprising, since similar ceiling effects have been observed in this system already at 4.5 years of disease duration20. Limited usability of motor-phenotypes in mid- and late-stage PD was also indicated for the re-examined patients of the present study where PD milestones were only reached for individuals with PIGD motor-phenotype at inclusion or at re-examination (data not shown), as shown in an earlier study29.For the motor-phenotype system we found no significant risk-stratification in adjusted analyses, and conclude that age and duration had a greater impact than the motor-phenotypes at the disease stages we studied, in concurrence with previous findings  higher PIGD score than others in this cohort, but similar onset age, sex, and duration, had 56.1, 100.3, and 32.5% higher risk for living at a nursing home, developing HY5, and dying during the observation period, respectively. These results support those of another study in which PIGD score but not tremor was associated with negative patient outcome23. In the present work, similar effects were indicated by Cox regression results  consisting of patients with PD living in three municipalities in southern Sweden  and patients with familial PD without known genetic cause on testing known to the Department of Neurology at Skåne University Hospital, Lund (hereditary subgroup). For the population subgroup, every resident in three adjacent municipalities  were contacted who had a diagnosis of PD or parkinsonism in registries from all public health care providers in the region between 2006 and 2010, and 76% of those contacted were included in the cohort.Patients within PARLU with PD or PD-dementia and complete baseline visits were selected for this study. Aiming for long-term observation, patients with <2 years of follow-up data were excluded. Patients whose diagnosis had been changed to any other disorder than PD or PD-dementia were excluded, as were two individuals with monogenetic disease Fig. .Fig. 3Fl38, modified HY-stage39, NMSQ40, and clinical assessment for other neurological symptoms. The presence of bradykinesia and one of rigidity, tremor, or postural imbalance was confirmed.Standardized baseline visits were performed from 2007 to 2013. Patients were then interviewed and clinically examined by the same physician (AP) including UPDRSIn 2017–2018, all surviving patients were invited to a follow-up research visit, including an interview and neurological re-examination by one physician (EYR). The same examination protocol was used with the addition of ACER. Patients used their regular medication on both examinations. We did not measure the doses of dopaminergic therapy because we aimed at evaluating the real-life situation of the patients and because several outcomes of this study were independent of treatment.2 was extracted from medical records of all individuals: regularly using walker, living in a nursing home, developing HY5, or dementia. This was performed 2018–2019 by one physician (EYR) by searching all medical records from medicine/neurology departments and memory clinics in the southern health care region  in Sweden, ranging back up to six decades. Medical records from primary health care were acquired when data was missing or inconsistent. All available paper records were scrutinized manually in full length. Electronic records were digitally searched for phrases, words, or parts of words associated with the milestones, including several grammatical forms and common spelling mistakes. Pre-defined criteria for fulfilling disease milestones were used. Patients were considered to have developed dementia when obtaining a diagnosis of dementia not otherwise specified or PD-dementia, being prescribed acetylcholinesterase-inhibitors, or when being repeatedly and clearly described as having dementia in medical records. Disease onset was defined as the first notion of rest tremor or subjective PD motor symptoms. Time at diagnosis was used when no description of onset was available (n = 6). To decrease the effect of peri-mortal comorbidities, milestones were ignored if they were only reached within two months before death. Periodicity of follow-up differed between patients and clinics, and to mitigate effects related to the exact timing of the patients’ contact with the medical services , all dates when reaching milestones were registered as a calendar year. Dates of birth, death, and end of observation were not standardized but registered as the actual date. The date of death was retrieved in 2019 from the Swedish population register kept by the Swedish Tax Agency, Skatteverket.Time to relevant social and clinical milestonesWritten informed consent was obtained from all included patients. If the patient was unable or incapable to decide, a close relative was instructed to determine patient consent and to act within the presumed previous intention of the patient. All parts of this study were approved by the Regional Ethics Review Board in Lund.4. The three motor-nonmotor subtype groups; mild-motor-predominant, intermediate, and diffuse-malignant, were determined by combining a composite motor score and three nonmotor parameters; a nonmotor rating scale, cognitive assessments, and assessment of REM-sleep behavioral disorder (RBD), similar to the original work3. We adapted the nonmotor parameters from the original publication to similar parameters collected in our study  as previously described4. In our cohort, 75th percentile cutoffs for motor-nonmotor categorization at baseline were 130.4 for composite motor score and 13.5 for NMSQ score. At re-examination, the simplified formula for composite motor score derived at baseline was utilized, but all other aspects of the systems, including cutoffs of the motor-nonmotor subparts (14.8 for NMSQ and 248.7 for composite motor score), was adapted to reexamination data.Of note, the cutoffs for the motor-phenotypes are absolute, whereas the classification in the motor-nonmotor subtypes is per design relative to the distribution of values in the entire cohort studied42. Adjustments also included disease duration since durations at baseline differed between patients. In each classification system, fulfillment of the five milestones was assessed with Kaplan–Meier survival curves, log-rank tests, and Cox regressions. The group with a worse prognosis in both classification systems was selected as the reference category in Cox regressions. The proportionality of hazards assumption was tested with the cox.zph command of the survival package in R v4.0.2. For all other statistical analyses, SPSS v25.0 was used. In the case of non-proportional hazards, analyses were instead performed in subgroups based on the least contributing covariate (sex), which made hazards proportional (Supplementary Table P values < 0.05 were regarded as significant and 95% confidence intervals were consistently used.Of 141 patients, eight individuals with > 20% of total data points missing in UPDRS parts II and III were excluded Fig. . For 28 Further information on research design is available in the Supplementary InformationReporting Summary


Herein, we define two factors affecting the DVS-BCB bonding quality through the DVS-BCB bonding mechanism. For strong DVS-BCB bonding, void-free and high-density chemical bonds are required. Therefore, we observed the DVS-BCB bonding under various compressive pressure conditions at a relatively low temperature (250 °C). The presence of voids and high-density crosslinking density was examined through near-infrared confocal laser microscopy and Fourier-transform infrared microscopy. We also evaluated the adhesion of the DVS-BCB bonding, using a universal testing machine. The results suggest that the good adhesion with no voids and high crosslinking density was obtained at the compressive pressure condition of 0.6 N/mm2. We believe that the proposed process will be of great significance for applications in semiconductor and device packaging technologies.Divinylsiloxane-bis-benzocyclobutene (DVS-BCB) has attracted significant attention as an intermediate bonding material, owing to its excellent properties. However, its applications are limited, due to damage to peripheral devices at high curing temperatures and unoptimized compressive pressure. Therefore, it is necessary to explore the compressive pressure condition for DVS-BCB bonding. This study demonstrates an optimization process for void-free DVS-BCB bonding. The process for obtaining void-free DVS-BCB bonding is a vacuum condition of 0.03 Torr, compressive pressure of 0.6 N/mm Owing to the development of the semiconductor industry, bonding wafers with complex structures of semiconductors and micro-electromechanical systems (MEMS) devices without interconnection delay, low adhesive strength, crosstalk, and power dissipation have attracted significant attention ,2. In pa2 plasma activation   group and three acetoxy (OCOCH3) groups [For DVS-BCB bonding with strong adhesion, it is necessary to understand the chemical reaction and chemical bonding of the materials used and to minimize the defects that may occur. In ) groups . The ace) groups . In gene) groups ,24,30. I) groups ,32,33,342O in the AP 3000 layer must be removed from the DVS-BCB layer. Second, it is necessary to create a high-density polymer with ideal chemical bonding of each material at an optimized curing temperature and compression process. We proposed an optimal void-free DVS-BCB bonding process by investigating the DVS-BCB bonding products manufactured, according to the compression conditions based on these two factors. We defined two factors that affect the strength of DVS-BCB bonding through chemical reactions that occur during the bonding process of DVS-BCB. First, all voids created by the vaporization of HWe measured optical microscopy images, as shown in 2, confirming that the two DVS-BCB silicon wafers are well aligned and bonded. In addition, it was confirmed that the voids generated by the water of the AP 3000 layer did not fall out of the DVS-BCB layer and still existed since the compressive pressure was lower than 0.6 N/mm2. In particular, when the compressive pressure was 0.4 N/mm2, it was confirmed that a large number of voids exist between the patterns, which is a path through which it is difficult for the pores to escape. When the compressive pressure was 0.5 N/mm2, several voids escaped outside the DVS-BCB layer, but it was confirmed that some voids were still present at some points. However, it was confirmed that void-free DVS-BCB bonding can be obtained at a compressive pressure of 0.6 N/mm2. Because DVS-BCB has high fluidity at high temperatures, voids are released to the outside of the layer through ideal compression under vacuum conditions. If these voids remain, they affect the bonding strength of the DVS-BCB bonding and eventually lead to cleavages. From the near-infrared confocal laser microscopy images obtained in 2. 2 without voids. Therefore, we found that the optimal void-free DVS-BCB bonding condition is 0.6 N/mm2, wherein voids do not exist and the pattern shape and DVS-BCB layer thickness do not deform.-1, which is the core fingerprint region of the molecule. Owing to the relatively large DVS-BCB molecule, there are numerous partially overlapping peaks in the fingerprint region [3 stretch (≈1255 cm−1), Si–O–Si stretch (≈1040 cm−1), Si–O bending (≈830 cm−1), and Si–CH3 bending (≈780 cm−1), which is consistent with the results of typical DVS-BCB bonding [3 bonding. The analysis confirmed that the absorption peak increased as the compressive pressure increased from 0.4 N/mm2 to 0.6 N/mm2. The increase in the absorption peak intensity indicates an increase in the Si–O–Si and Si–CH3 bond densities based on the same area, thereby suggesting that more uniform and more bridged bonds were generated. These results are directly related to the adhesive strength of DVS-BCB bonding, and it is expected that the strongest and most stable bonding is achieved at 0.6 N/mm2. We report the importance of the degree of chemical bonding in DVS-BCB bonding, in addition to the presence or absence of voids, as a factor that significantly affects the adhesion strength of DVS-BCB bonding.We conducted an FT-IR analysis to determine the effects of various compressive pressure conditions on the chemical bonding behavior in the DVS-BCB bonding process. For accurate comparison of the samples, a portion with the same thickness was found in the exposed DVS-BCB layer through precise cutting, and it was measured, using an FT-IR microscope (spot size 80 μm × 80 μm). As shown in t region . As show bonding ,37. With2) epoxy. We used epoxy with high adhesion strength to cut the DVS-BCB layer and confirmed that the DVS-BCB bonding layer was detached, as shown in 2 at compressive pressures of 0.4, 0.5, and 0.6 N/mm2, respectively. These results are consistent with the results of 2. The adhesive strength at 0.4 N/mm2 is lower than those at 0.5 and 0.6 N/mm2, owing to the presence of a large number of voids and low chemical bonding. From the results of 0.5 and 0.6 N/mm2, it can be seen that the increase in the adhesion strength line shape with respect to the displacement is similar. Furthermore, it was confirmed that the compressive pressure of 0.6 N/mm2 resulted in greater adhesion strength by 0.37 N/cm2 than the compressive pressure of 0.5 N/mm2; this phenomenon can be attributed to the difference in the number of chemical bonds rather than the presence of voids generated during the DVS-BCB bonding process. Based on these results, we confirmed that the morphological, chemical, and physical analysis results of the DVS-BCB bonding are in good agreement. In the DVS-BCB bonding process, the high compressive pressure induces high stress on the high-density pattern array. These high stresses can destroy or crack the high-density pattern array structure. Therefore, we propose that the optimized compressive pressure condition for void-free DVS-BCB bonding is 0.6 N/mm2. We carried out near-infrared confocal laser microscopy to examine the presence of voids even in DVS-BCB bonding with different patterns under the proposed compressive pressure condition of 0.6 N/mm2. It was confirmed that there was no void in the super high-density patterns and linear patterns, as shown in We confirmed the presence of voids and the behavior of chemical bonds in the DVS-BCB bonding fabricated according to the compressive pressure through near-infrared confocal laser microscopy and FT-IR analysis. We conducted a tensile test using a universal testing machine to investigate the effect of these properties on the adhesive strength of the DVS-BCB bonding. In the tensile test, an aluminum jig with dimension of 12.5 mm × 32 mm × 14.5 mm (w × l × h) and the prepared sample were strongly attached, using AXIA EP-04 (tensile shear strength of 2962 N/cm2 has the best adhesion, highest chemical bonding density, and no voids. Moreover, it is confirmed that the 3D structure of the DVS-BCB layer is not destroyed and the thickness of the DVS-BCB bonding layer is not deformed, thereby indicating no deformation of the DVS-BCB bonding layer by the compressive pressure. Based on our results, we found that the optimized void-free DVS-BCB bonding process is temperature curing at 250 °C for 1 h at a compressive pressure of 0.6 N/mm2 under vacuum conditions of 0.03 Torr. We expect that this proposed void-free DVS-BCB bonding process can be applied to the bonding and packaging processes of high-density MEMS device arrays, 3D integrated circuit devices, optical devices, and MEMS.In this study, the shape and physicochemical properties of DVS-BCB bonding according to the compressive pressure were investigated to determine the optimal void-free DVS-BCB bonding process conditions. The optimal process conditions result in good adhesion and void-free bonding, even in a narrow area such as a high-density pattern array. We propose the presence of voids and differences in crosslinking density as factors affecting DVS-BCB bonding through the bonding mechanism of DVS-BCB bonding. Through near-infrared confocal laser microscopy, FT-IR, and universal testing machine analysis, we found that the presence of voids and the crosslinking density of the materials have a significant effect on the bonding strength of the DVS-BCB bonds. We confirmed that the DVS-BCB bonding sample produced with a compression strength of 0.6 N/mm


Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM−1cm−2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from  A (bio)electrochemical detection and conversion of various chemical compounds is a rapidly evolving approach, which requires novel electrodes and optimized methods for immobilization of biocatalysts, particularly enzymes . An effiGraphene oxide- or graphite-based electrocatalytic systems attract attention due to many anticipated properties including morphology, surface chemistry and electrical characteristics . Gold na−1 caloric value, which is 38% of the energy content of sucrose, it can be used as a natural low-calorie bulk sweetener [Sterculia setigera gum and various processed foods , but its availability appears limited and its recovery is expensive, therefore creating a major impediment to its wider use in industry [Valorization of biomass by using electrochemical processes is becoming increasingly raised and numerous by-products are produced by conversion of agro-wastes. Hence, D-galactose can be obtained via lactose hydrolysis in the dairy industry . Also, Dweetener ,16,17. Iindustry . To overindustry  and a biindustry ,21,22 haindustry . In contindustry ,25,26,27Obviously, evaluation of the total amount of D-tagatose in industrial products is very important for their quality; also it is very important for D-tagatose biosynthesis monitoring. At present, only several methods are suggested for D-tagatose determination: the colorimetric method , mass spGluconobacter industrius (FDH) was chosen and tested as a D-tagatose recognition element in this work. FDH catalyzes oxidation of D-fructose to 5-keto-D-fructose, but lately it has been shown that the FDH immobilized on nanoporous gold can weakly oxidize other sugars and polyols such as D-glucose, D-galactose and D-mannitol (the highest response of 5% was obtained with glucose) [+/2e− oxidation to 5-keto-D-fructose; subunit II, which acts as a built-in electron acceptor with three heme c moieties covalently bound to the enzyme scaffold with two of them involved in the stepwise electron transfer pathway; subunit III, which is not involved in the electron transfer, but plays a key role for the enzyme complex stability [While D-tagatose is ketohexose and has a structure similar to D-fructose, except for the orientation of the hydroxyl group on C4 ,35, D-frglucose) . FDH is tability ,38. A natability . Moreovetability . Second,tability ,41; henctability . Severaltability .To combine a mediator-free DET approach with modulation of FDH substrate specificity via immobilization on different surfaces, three different electrode nanomaterials with immobilized FDH were used to design three types of biosensors: (i) monolayer of gold nanoparticles (AuNPs) coated with 4-mercaptobenzoic acid (4-MBA) (AuNP/4-MBA/FDH), (ii) AuNPs coated with 4-aminothiophenol (PATP) (AuNP/PATP/FDH) and (iii) a layer of TRGO on graphite (TRGO/FDH). The sensitivity and selectivity of those three systems to D-tagatose and their operational stability were investigated. The TRGO/FDH was also tested in a bioreactor that mimicked bioconversion technology in which by-product D-galactose was converted into D-tagatose by employing L-arabinose isomerase (L-AI).Gluconobacter industrius  was purchased from Sigma-Aldrich . The recombinant thermophilic L-arabinose isomerase (L-AI) (5.6 U·mg−1) from Geobacillus thermoleovorans DSM 15325 was prepared as described previously [4⋅3H2O and trisodium citrate according to the Turkevich synthesis method [D-Fructose dehydrogenase (EC 1.1.99.11) from eviously . The fireviously . Gold nas method . The cons method . Graphit2 in 50 mM PBS (pH 7.5). Unless stated otherwise, all reactions were performed at 50 °C for 5 min. The generated D-tagatose was determined by the cysteine carbazole sulfuric-acid method, and the absorbance was measured at 560 nm [L-AI activity was measured by determination of the amount of D-tagatose. Each reaction mixture contained 100 mM D-galactose and 1 mM MgClt 560 nm . One unit 560 nm .−1) in the McIlvaine buffer solution (pH 4.5) . Then the biosensor was placed under a glutaro-aldehyde vapor condition at 20 °C for 30 min. Finally, the biosensor was designed by mechanically attaching and fixing the flexible terylene film with a rubber ring to the pretreated surface of the electrode. The basic scheme of the biosensor construction is presented in Aiming to design D-tagatose biosensor, TRGO was extruded by forming a tablet. The tablet was sealed in a Teflon tube with amorphous carbon pasta. Electrodes were washed with deionized water (DI), and dried before use. A biosensor was prepared by the adsorption on the TRGO surface of 2 µL, 0.5% triton X-100 solution in water  and 2 µL of FDH  were placed on the cleaned gold electrode surface and allowed drying at room temperature. Subsequently, the electrode was electrochemically cleaned (30 CV scans in 0.5 M H2SO4) and submerged in a 5 mM 4-mercaptobenzoic acid or 4-aminothiophenol solution in methanol and left overnight. Afterwards, the electrodes were thoroughly rinsed with deionized water and 2 μL of FDH (1471 U mL−1) in a McIlvaine buffer solution (pH 4.5) was placed and left for 30 min at 10 °C. The basic scheme of the biosensor construction is presented in Before the experiments, gold electrodes were polished with aluminum oxide slurry (0.3 μm), rinsed with deionized water and sonicated for 4 min in DI and 4 min in acetone to remove bounded particulates. After sonication, the working electrode was thoroughly washed with DI and treated by electrochemical cleaning. Briefly, 30 cyclic voltammetry (CV) scans were run from −0.2 to 1.75 V vs. Ag/AgCl and backwards in 0.5 M HAmperometry measurements were performed using an electrochemical system  with a conventional three-electrode system comprised of a platinum plate electrode as an auxiliary, a saturated Ag/AgCl electrode as a reference and the working electrode D-tagatose biosensor designed on a base of three electrode surfaces. The response of the prepared biosensors to the addition of enzyme substrate was investigated under potentiostatic conditions at 0.4 V in a stirred McIlvaine, pH 4.5, and PBS, pH 7.5, buffer solutions. All measurements were obtained at 20 °C temperature.j) on substrate concentration (C) the apparent Michaelis constant values (maxj) were calculated. For this, the response current density was measured three times in the solution with C and the average response j was obtained. The experimental dependence j vs. C was approximated by OriginPro 8  according to the electrochemical version of the Michaelis–Menten equation [From the experimental dependence of the current density  at 50 °C in a stirred PBS (pH = 7.5) containing 444.4 mM D-galactose. L-AI was kept in a dialysis bag in the center of the reactor  were taken every 10 h until 50 h to evaluate the concentrations of synthesized D-tagatose.3 powder with 120 μL of distilled water. 10 μL of the suspension were dropped onto a silica plate and dried under 110 °C for 10 min. Then samples were left in a ventilating hood until the sample temperature decreased to 30 °C. Before each measurement the samples were additionally dried under 50 °C for 20 min. The aqueous suspension of the AuNPs were put on a gold disk electrode, which was cleaned as described previously and dried under a nitrogen stream at room temperature (RT). Imaging was performed in the air at RT.TRGO and AuNP were analyzed by scanning probe microscope . The tapping mode of surface scanning was used for visualization and characterization. The data and AFM images were processed by the NanoScope Software 6.14 (Veeco Instruments Inc.). The aqueous suspension of TRGO was obtained by mixing of 0.5 mmFDH can act as a DET-type enzyme in D-fructose bioelectrocatytic oxidation ,49; howeThe AFM data clearly show that TRGO is composed of nanometric particles. The measured average diameter of the TRGO particles was ∼11 nm and the average particle height was ∼0.5 nm, which is very close to that of the single layer of graphene (0.39 nm).2) and the measured surface area (3.55 μm2). In addition, AFM measurements revealed the size of AuNPs to be ∼19 nm; the size of AuNP was obtained using a ten-fold diluted nanoparticle solution. Using AFM data, it was found that AuNPs increase the surface area by 50 percent. This difference was calculated by comparing the difference between the geometrically flat surface  AuNP/4-MBA, nor for AuNP/PATP or TRGO after addition of D-tagatose or D-fructose (data not shown). In contrast, the CVs obtained on three types of electrodes with the enzyme exhibited a bioelectrocatalytic current . While tAs can be seen in Previous studies ,51,52 coAiming to test the ability of FDH to catalyze the oxidation of D-tagatose to 5-keto-D-tagatose, FDH was immobilized onto electrode surfaces under experimental conditions and chronoamperometric measurements were performed. The responses of the manufactured biosensors to D-tagatose and D-fructose were recorded as a difference between the steady-state current and the background current. Conversion of both substrates by FDH immobilized on three tested electrode surfaces was observed. Taking into account the previous studies ,38, it wmaxj(D-tagatose)/maxj(D-fructose) ∗ 100% where maxj(D-tagatose) and maxj(D-fructose) are maximal current densities that can be generated by the bioelectrocatalytic system. maxj for both substrates were theoretically calculated using calibration graphs (The sensitivities of the biosensors were obtained from the slope of a linear relationship between current density and D-tagatose or D-fructose concentration presented in n graphs  and the n graphs  demonstrAccording to data in equation . These v−1 cm−2) and proper stability (data not shown).The lowest value of nto TRGO . The bioIn fact, the effective DET requires a proper spatial orientation of the enzyme, which should be located a short distance to the electrode surface in the way that the subunit II (the heme c-domain) of FDH should be facing toward the electrode surface. Recent research has shown that depending on the positive/negative surface charge, hydrophobicity/hydrophilicity determines the proper orientation of the enzyme , but thiSince the biosensor TRGO/FDH exhibited the best DET results, it was decided to employ this biosensor for further research in the reactor for the bioconversion of D-galactose into D-tagatose.In order to study a possibility of the application of the biosensor in real media, a prototype bioreactor was designed . As the The selectivity of the prepared biosensor was further studied to evaluate the influence of D-galactose on the determination of D-tagatose. As can be seen in Previously we showed that an amperometric biosensor based on TRGO and immobilized FDH displayed an appropriate long-term stability, hence, after a period of five days the biosensor sensitivity remained more than 80% of the initial response . In thisAs can be seen in Aiming to demonstrate practical applicability of the proposed biosensors, the TRGO/FDH biosensor was employed for the quantification of D-tagatose in the prototype bioreactor . This reBased on data presented in the The accuracy of the amperometric biosensor was confirmed by plotting the results obtained by the amperometric biosensor vs. the results obtained using spectrophotometric analysis .xy) between two independent analyses: spectrophotometric and by the TRGO/FDH biosensor of the bioconversion mixture was 0.9974. This indicated an excellent agreement between the two methods. The slope of the correlation straight was of 0.9978, which indicates, that results, determined using the amperometric biosensor, were slightly lower than those of spectrophotometric analysis. These findings suggest that, in general, the TRGO/FDH biosensor generated a correct response to D-tagatose in the D-galactose/D-tagatose mixture.The correlation coefficient  coated with 4-mercaptobenzoic acid (AuNP/4-MBA) or 4-aminothiophenol (AuNP/PATP) monolayer, and a layer of thermally reduced graphene oxide (TRGO) on graphite.Because native FDH does not catalyze D-tagatose oxidation in bulk solutions, it can be concluded that oriented immobilization of the enzyme onto the proposed electrode surfaces modifies FDH’s selectivity towards D-tagatose. Different values of specificity to D-tagatose for all three biosensors revealed that immobilization of FDH on these surfaces remained unique and herewith led to slightly different conformations of the 3-dimensional form of FDH. Notwithstanding that a bioelectrochemical response towards D-tagatose is significantly lower comparing to the response to common substrate—D-fructose, the developed biosensors are entirely applicable to monitor a formation of D-tagatose during the isomerization process of D-galactose.This research demonstrated that a specificity of FDH to D-tagatose can be changed using proper electrode materials for immobilization of the enzyme. We propose that during immobilization, FDH undergoes conformational changes as it binds to the electrode surface, resulting in the proper spatial orientation required for direct D-tagatose electrocatalytic oxidation. This assumption was confirmed by calculated values of the apparent Michaelis constant (As TRGO/FDH demonstrated the highest sensitivity to D-tagatose, it was chosen to investigate the biosensors’ applicability in a prototype bioreactor. Independent spectrophotometric analysis of the bioconversion mixture revealed that the biosensor was suitable for monitoring of D-tagatose during a bioconversion of D-galactose by L-arabinose isomerase. Because the biosensor showed no response in the presence of various quantities of D-galactose, it may be concluded that D-galactose is not a substrate for FDH and hence has no effect on the biosensor’s response.While a simple approach for monitoring D-tagatose in industrial projects is still in great demand, the proposed electrochemical biosensors could address that void. Because there is currently no enzyme for selective D-tagatose oxidation that could serve as a recognition element for electrochemical biosensors, we propose an alternative—FDH with adjusted D-tagatose selectivity. Such a biotechnological solution could be very promising for development into a process for valorizing dairy industry waste.


Viridibacillus sp. The produced G-AgNPs were characterized by various analytical methods, including UV-Vis spectroscopy, single-particle inductively coupled plasma-mass spectrometry (sp-ICP-MS), scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDX), elemental mapping, transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and Thermogravimetric analysis (TGA). The reduction of Ag+ to Ag° was observed by UV-Vis spectroscopy, which demonstrated the formation of stable G-AgNPs with a Surface Plasmon Resonance (SPR) band at the maximum of 430 nm. TEM analysis demonstrated that the G-AgNPs were spherical with a 5–30 nm size range. The produced G-AgNPs were stable for more than 1 year in an aqueous solution at 4°C. Importantly, G-AgNPs showed remarkable antimicrobial activity against Gram-negative pathogens- E. coli and P. aeruginosa with MIC values of 0.1 and 4 μg/mL and MBC values of 1 and 8 μg/mL, respectively. This level of antimicrobial activity is superior to other AgNPs reported in the literature.Recently, green silver nanoparticles (G-AgNPs) have gained much attention in medical science due to their extraordinary effects against multidrug-resistant microorganisms. The strong antimicrobial nature of G-AgNPs corresponds to their unique physicochemical properties such as size, shape, surface charge, and active surface groups available to interact with the pathogens. The current study demonstrates a simple, environmentally friendly, and economical method to produce G-AgNPs from an environmental isolate of  Silver nanoparticles (AgNPs) are widely known for their industrial applications in the field of medicine, pharmacology, food, agriculture, cosmetics, and textiles due to their unique antimicrobial properties, which further depend on nanoparticles (NPs) structure . The mosCamellia sinensis and Prunus Africana. The current study deals with the extracellular synthesis of G-AgNPs from an environmental isolate without any additives for the reduction or stabilization process. The extracellular constituents of cells act as reducing and stabilizing agents. In addition to extensive characterization, we explored the synthesized G-AgNPs against E. coli, and P. aeruginosa, to study their antibacterial property.Developing resistance mechanisms in pathogenic microorganisms against current and developing drugs has become a prime concern in the medical field. Understanding the developing resistance mechanisms in these pathogens is important, and designing novel and strong antimicrobial agents that can overcome or circumvent the resistance is equally important . Indeed,3), tryptic soya agar (TSA), tryptic soya broth (TSB), and Luria broth (LB) were purchased from Sigma-Aldrich Chemicals, St. Louis, MO, United States.Silver nitrate  field, Lyngby, Denmark. Single colonies were isolated by using the serial dilution technique on TSA plates. All the isolates were tested for primary AgNPs production, and the strongest strain was chosen for further studies. Molecular identification of the isolated potential strain was performed using 16S rDNA amplification and sequencing. Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) and used as a template for PCR with the universal primers 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492 R (3′-TACGGYTACCTTGTTACGACTT-5′) . Eurofin3 and incubated in a shake flask incubator at 37°C, 200 rpm, and 24–48 h. The silver salt mix supernatant (reaction medium) was monitored continuously for AgNPs production, by visual inspection, and by recording the UV-Vis spectra of the reaction medium at definite time intervals. Once the G-AgNPs were formed, for purification, the reaction medium was centrifuged at 3,000 for 5 min to remove any big and unwanted components. Then the same medium was centrifuged at 14,000 rpm for 15 min  to G-AgNPs was initially monitored To know the concentration of produced G-AgNPs, sp-ICP-MS  was performed. The stability of G-AgNPs was examined by using the purified G-AgNPs and keeping them for a different time, temperatures, and indifferent bacteriological media such as TSB and LB. The results were observed by visible inspection of which pictures are taken, UV-Vis, and sp-ICP-MS analysis before and after the defined period .Thermogravimetric analysis  was performed to check the temperature stability G-AgNPs. For analysis, G-AgNPs samples in dried and powdered form were placed in an alumina pan and heated from 20 to 700°C at a ramping time of 10°C/min.Scanning electron microscopy examination with energy dispersive X-ray (EDX) and elemental mapping was performed to study the G-AgNPs morphology and elemental composition. EDX analysis setup was coupled with the SEM instrument. Sample preparation was done by dropping 5 μl of pure G-AgNPs (0.1 mg/mL) on carbon tape and air-dried at room temperature (RT) for 15 min. SEM Micrographs were recorded using a Quanta FEG 200 ESEM microscope .Transmission electron microscopy study using FEI Tecnai T20 G2 was conducted to analyze the internal morphology, composition, and crystallographic information of G-AgNPs. The instrument was operated at an acceleration voltage of 200 kV. A sample of G-AgNPs was prepared by spotting a drop of pure NPs solution suspended in water on a carbon-coated copper grid. The sample-containing grid was completely dried before analysis.1 measurements were carried out in intermittent contact mode using standard probes of single-crystal highly doped silicon with a radius of curvature of less than 30 nm (SuperSharpSiliconTM Non-contact AFM probes from Nanosensors). The standard uncertainty u(d) of the measured diameters is u(d) < 0.05 day (Atomic force microscopy (Park NX20)0.05 day .Dynamic light sattering measurements were performed to study the size distribution concerning intensity and zeta potential of pure G-AgNPs. Particle size measurement was executed using Zetasizer Nano ZS, Chuo-ku Kobe-shi, Japan. The autocorrelation functions of the samples were analyzed using the Contin algorithm through the Zetasizer 7.12 software. Samples were run in triplicates .–1. The recorded spectra recorded were plotted as transmittance (%) vs. wavenumber (cm–1).Green silver nanoparticles were subjected to Fourier transform infrared (FTIR) analysis to determine the presence of biomolecules, functional groups responsible for the reduction and capping/stabilization. The FTIR measurements were carried out using Nicolet iS50  by scanning the air-dried purified G-AgNPs and freeze-dried cell’s supernatant within the range of 500–4,000 cmEscherichia coli UTI 89, and Pseudomonas aeruginosa PAO1. Both the strains were grown overnight in LB medium at 37°C for 24 h. The overnight grown cultures were diluted to approximately 1–2 × 105 colony-forming units (CFU)/mL using LB medium. Then, the G-AgNPs were added in concentrations ranging from 0.1 to 16 μg/mL. The LB medium containing respective pathogenic bacteria and G-AgNPs were further incubated in a shake flask incubator at 37°C, 150 rpm, for 24 h. After 24 h, the samples were analyzed by measuring the optical density (OD) at 550 nm. The MIC was defined as the lowest concentration of G-AgNPs, which inhibited the bacterial growth, measured as OD550. The MBC value was defined as the lowest concentration of G-AgNPs required to kill the respective bacterial strain. To measure MBC, 100 μL of the LB medium containing respective pathogenic bacteria and G-AgNPs were spread on agar plates and incubated at 37°C overnight, followed by a CFU count.The antimicrobial activity of G-AgNPs was evaluated against two Gram-negative pathogens: To visualize the viable and dead cells, control cells and cells treated with G-AgNPs were stained for 20 min with a mixture of 6.0 μM SYTO 9 and 30 μM KI from Live/Dead BacLight Viability kit L13152 . Fluorescence microscopic imaging of the cells was performed using a LEICA DM 4000 B .To evaluate the drastic effects of G-AgNPs on individual cells, SEM was carried out. SEM was performed by fixing the control and treated cells with 3% of glutaraldehyde overnight at 4°C. The next day, the samples were dehydrated with graded series of ethanol concentrations  for 15 min and with absolute ethanol for 20 min. The dehydrated samples were placed on SEM carbon tape and left to dry at RT. The samples were coated with gold before SEM imaging. EDX and elemental mapping of G-AgNPs treated cells was also performed to check that the killing effects are due to the action of G-AgNPs only.Viridibacillus arvi strain LMG 22165. The isolated strain sequence number is submitted to NCBI with GenBank ID: SUB10641455. Viridibacillus arvi is reported to be Gram-positive, aerobic, spore-forming, rod-shaped bacteria  property of G-AgNPs . We then examined the stability of G-AgNPs over short and long periods. The measurements were made after 24, 48, 86 h, and 1 year . Our stuvia EDX spectroscopy, which reveals the presence of the strong elemental signal from silver at 3 keV  diffraction (SAED) studies. SEM image examination showed the spherical structure of G-AgNPs . The eleat 3 keV . The cor 5–30 nm . However 5–30 nm . These v 5–30 nm ; they su 5–30 nm . Hydrody 5–30 nm . The zet 5–30 nm , which s– or carboxylate groups), 561.08 which are identical to the supernatant spectrum. The FTIR results indicate the presence of carboxyl groups (-C=O), and amine groups (−NH) which represents the presence of proteins, amino acids, and other biomolecules originating from the supernatant on the surface of the produced G-AgNPs, responsible for capping and stabilizing G-AgNPs  against E. coli, i.e., 8 μg/mL, in our study. Similarly, Abelmoschus esculentus. The authors showed the MBC value of AgNPs against E. coli and P. aeruginosa were 110 and 105 μg/mL  can be found in the article/supplementary material.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.PS designed and performed the experiments, analyzed the results, and prepared the manuscript and figures. IM supervised all experimental work and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, we report a novel transient bio-fluorochromic supramolecular co-assembly mediated by a hydrolytic enzyme  towards rewritable security printing. A co-assembly based on the designed tetrabranched cationic diethynylanthracene monomer tends to be formed by adding adenosine triphosphate (ATP) as the biofuel. The resulting co-assembly possesses a time-encoded bio-fluorochromic feature, upon successively hydrolyzing ATP with ALP and re-adding new batches of ATP. On this basis, the dynamic fluorescent properties of this time-encoded co-assembly system have been successfully enabled in rewritable security patterns  Rewritable security printing has been successfully achieved based on a biofuel-driven transient supramolecular co-assembly mediated by an enzyme, providing fascinating potential for artificial functional materials with a biomimetic mode. Additionally, the printed information is usually not erasable, and the paper can only be used once. Hence, it is highly desirable to develop advanced fluorescent materials with security printing and rewritable features.Paper document security is of paramount importance even in the electronic information world, because the widespread medium for documentation is still paper.16–19 However, the disadvantage of this strategy is that rare responsive units are available. An ingenious approach is to dynamically regulate the packing mode of π-conjugated molecules in a noncovalent self-assembly way, resulting in a reversible change of fluorescence intensity and wavelength.20–23 In this respect, fluorochromic supramolecular assemblies triggered by heat, light, solvent, vapor and force have drawn great enthusiasm.24–28 Among them, bio-fluorochromic assembly is of particular interest, because of its possible transient characteristics in response to biological sources, which can be regarded as a superior candidate for smart fluorescent materials. Nevertheless, reports highlighting bio-fluorochromic assembly and biofueled transient assembly29–34 in one system have been rarely exploited so far, but need to be addressed because they are closer to the naturally occurring system and more efficient in some specific scenarios. Here, we propose a simple co-assembly strategy to build a novel transient bio-fluorochromic system, where the emission signals of π-conjugated units can be reversibly modulated on a time scale, nicely meeting the color variability and repeatability requirements for exploiting in rewritable security printing.To attain these objectives, a feasible strategy is to exploit various fluorophores with stimuli-responsiveness by chemical synthesis.1 has been firstly designed and synthesized , which is similar to the naturally occurring self-assembled cytoskeleton proteins in living cells.35,36 Thus, time-encoded fluorescence is generated in the synthetic assembled system, via repeatedly adding new batches of ATP anthracene, the core of 1, possesses red-shifted fluorescence in the aggregated states, which is suitable for being employed to construct fluorochromic materials.37,38 Detailed synthetic procedures and characterization results are shown in the ESI.†The structure of desired monomer 1 is carefully investigated. In dilute aqueous solution, the UV-Vis spectra of 1 show two major absorption bands in the regions of 300–325 nm and 350–500 nm with vibronic fine structures, indicative of molecularly dissolved states  at around 460 nm gradually decreases, while a red-shifted shoulder band located between 470 and 530 nm appears  is 2.4 × 10−5 M  display two isosbestic points (452 and 469 nm) upon heating  versus temperature, a sigmoidal melting curve is obtained  value of −87.5 kJ mol−1, together with a Tm (melting temperature at which αagg is 0.5) value of 306.2 K (Table S1G (Gibbs free energy) value of the self-assembly process of 1 is calculated to be −24.9 kJ mol−1 at 298 K , we then sought to investigate the co-assembly of 1 with ATP. Intriguingly, as ATP is progressively introduced into the solution of 1 (2.0 × 10−5 M),47 the original absorption bands located between 350 and 550 nm gradually decrease, while a new set of bands centered at 457 nm emerges with an isosbestic point at 479 nm .48 Detailed titration experiments reveal that around one equivalent of ATP is required to bind with 1 , the resonances on 1 and ATP shift up-field , while the well-defined signals become broad  is then considered to reveal the gradual evolution of Cotton effects of 1/ATP. The CD signals progressively reach a plateau with an isodichroic point at 383 nm  signal can be detected for 1/ATP  simulations are performed. The simulation box of the co-assembly 1/ATP containing 14 monomers 1 and 14 ATP molecules in explicit water solvent is calculated based on a CHARMM36 force field at 298 K for 20 ns to equilibrate the initial configuration anthracene planar cores and the chiral adenosine lying at the periphery of the aggregates, through the intermolecular electrostatic and hydrogen-bonding interactions between the R–NH3+ units in 1 and phosphates in ATP. All three of the phosphates in ATP participate in the binding with 1  or monophosphate (AMP: adenosine monophosphate) leads to a different co-assembly behavior from that of 1/ATP. Specifically, around two equivalents of ADP are required to totally change the emission and absorbance signals  under mild conditions.52 Inasmuch as the hydrolysates cannot induce 1 to assemble into co-assemblies, 1/ATP would disassemble along with the hydrolysis of ATP. When ALP is added to the co-assembly of 1/ATP, the intensity of fluorescence spectra gradually increases up to the initial intensity of that of monomer 1, indicative of the ALP induced disassembly and release of free monomers  shortens from 14.5 min via 7.3 min to 3.6 min (−1, 0.12 min−1, and 0.29 min−1. The results unambiguously prove that the rate of the enzymatic hydrolysis reaction for ATP directly reflects the progress of the disassembly process. Additionally, the rate of the disassembly process of 1/ATP could also be regulated by the temperature of enzymatic hydrolysis, which demonstrates a faster disassembly process at a higher temperature (Furthermore, the kinetics of the disassembly process of  3.6 min . The corperature .1/ATP is re-formed. More interestingly, the fluorescent signal reappears spontaneously with time increasing, which is ascribed to the presence of ALP in the solution system. Subsequently, with the addition of ATP again, the same trend as the previous cycle is observed. With continuously adding new batches of ATP, the emission signal can be reversibly switched for multiple cycles (1/ATP are confirmed at a different order of ALP units. Upon increasing the amount of prestored ALP in 1/ATP, the shortened half-life times of the transient cycles are observed from the time-dependent fluorescence spectra (With the re-addition of ATP into the solution mixture, the green emission color is immediately quenched, accompanied by fluorescence spectra disappearance . Meanwhie cycles . Further spectra . The sam spectra . Hence, 1/ATP can be fine-tuned with ALP, it can be employed for developing time-encoded bio-fluorochromic materials. To verify this assumption, a 3D model of fluorescence changing with time is fabricated firstly. In detail, 2 wt% agarose aqueous solution is heated to clarify, and then transferred into a 3D printed mold (1/ATP/ALP for several minutes. When the model is standing at room temperature, the green emission color gradually emerges within 20 min (1/ATP disassembles in the hydrogel along with the enzyme-catalyzed hydrolysis process of ATP.53 Thus, it is evident that the ALP mediated transient assembly of 1/ATP can be regarded as an effective strategy for the construction of time-encoded emission systems.Considering that the emission signals of ted mold . A non-fn 20 min . This sp1 is firstly filled into the customized cartridge of an inkjet printer (1 and ATP, resulting in the fluorescence quenching of the printed location on the paper. The QR code is invisible under natural light, but can be decoded under UV light by using a smartphone. Interestingly, the temporary QR code is gradually erased with time increasing, which fails to be recognized after 10 min. Such results are ascribed to the presence of ALP in the printed area, which destroyed the co-assembly 1/ATP by hydrolysing ATP. Thus, the ATP/ALP aqueous solution can be used as a transient ink to transfer messages with time-encoded features in security papers. Furthermore, upon reprinting with ATP/ALP inks, the writing/erasing process can be performed multiple times (1/ATP possesses temporal emission signals mediated by ALP on a time scale, which is suitable for multiple-time uses in security printing.On this basis, we then turn to explore its potential application in rewritable security printing. Specifically, the aqueous solution of  printer . A squarle times . Moreovele times . The rewle times . Overall1 and anionic biofuel ATP. The nonequilibrium state of co-assembly 1/ATP can be finely controlled in aqueous medium by temporally regulating the consumption of ATP using its hydrolytic enzyme ALP, accompanied by fluorescence signal variations. Furthermore, the resulting bio-fluorochromic system is exploited to achieve rewritable security printing, by taking advantage of its time-encoded multiple-cycle emission switching features. Hence, the present work not only opens up a new avenue towards artificial functional materials with a biomimetic mode, but also greatly improves the reusability and security level of paper-based confidential information.In summary, we have successfully established a transient bio-fluorochromic supramolecular system through the electrostatic and hydrogen bonding interactions between the tetrabranched cationic diethynylanthracene monomer Detailed synthetic procedures, analytical data, and computational methods are provided in the ESI.Z. G. and W. T. conceived the idea for this project. S. Q., F. Y. and S. Z. performed the experiments, analyzed the data, and produced the artwork under the direction of Z. G. and W. T. All authors contributed to the manuscript preparation.There are no conflicts to declare.SC-012-D1SC03105H-s001


Core/shell nanocrystals (NCs) integrate collaborative functionalization that would trigger advanced properties, such as high energy conversion efficiency, nonblinking emission, and spin–orbit coupling. Such prospects are highly correlated with the crystal structure of individual constituents. However, it is challenging to achieve novel phases in core/shell NCs, generally non-existing in bulk counterparts. Here, we present a fast and clean high-pressure approach to fabricate heterostructured core/shell MnSe/MnS NCs with a new phase that does not occur in their bulk counterparts. We determine the new phase as an orthorhombic MnP structure (B31 phase), with close-packed zigzagged arrangements within unit cells. Encapsulation of the solid MnSe nanorod with an MnS shell allows us to identify two separate phase transitions with recognizable diffraction patterns under high pressure, where the heterointerface effect regulates the wurtzite → rocksalt → B31 phase transitions of the core. First-principles calculations indicate that the B31 phase is thermodynamically stable under high pressure and can survive under ambient conditions owing to the synergistic effect of subtle enthalpy differences and large surface energy in nanomaterials. The ability to retain the new phase may open up the opportunity for future manipulation of electronic and magnetic properties in heterostructured nanostructures. Core/shell MnSe/MnS nanocrystals with the B31 phase are thermodynamically stable under high pressure and can survive under ambient conditions owing to the synergistic effect of subtle enthalpy differences and high surface energy in nanomaterials. To date, an array of classic semiconductors  and III–V materials have been used to fabricate 1D coaxial core–shell heterostructures.17–19 However, these core–shell heterostructures are usually composed of well-known conventional phases that are existed in the corresponding bulk counterparts.20 The search for 1D core/shell NCs with unusual crystal phases is essential for the development of novel phase-dependent properties and materials, yet their controllable synthesis and delicate modulation remain elusive.Heterostructured core/shell and heterojunction nanocrystals (NCs) have emerged as an interesting class of materials because of their unique optical, electronic, catalytic and magnetic properties originating from their individual constituents.21,22In situ pressure-processing has been considered as a fast and clean mechanical method for the fabrication of nanomaterials with a controlled morphology and phase without involvement of chemical reactions and post purification processes.23–25 Unlike solution-phase synthesis, the high-pressure technique allows for monitoring structure modulation of nanomaterials under continuous compression, offering direct evidence for the structural stability and transition process of materials.26 Inspired by the pressure-induced structural phase transition of CdSe nanoparticles, significant advances in high-pressure nanotechnology have been accomplished by tuning the morphology, construction and crystal structure.27–30 However, relatively few studies have been reported for high-pressure nanophases that could be anticipated to survive under ambient conditions, in particular unconventional high-pressure new phases.Pressure-induced phase engineering offers opportunities for the rational design and synthesis of materials with unusual crystal phases, in particular, unconventional high-pressure new phases.In this work, we undertake a study on the high-pressure phase transition behaviors of heterostructured core/shell MnSe/MnS nanorods, especially focusing on the new-phase retention engineering of core/shell nanostructures. The results of this study indicate that high pressure could trigger wurtzite (WZ) → rocksalt (RS) → B31 phase transitions in the core/shell MnSe/MnS nanorods by a combination of high-pressure angle dispersive X-ray diffraction (ADXRD) and high-resolution transmission electron microscopy (HRTEM) characterization studies as well as first-principles calculations. Furthermore, the generated new phase core/shell nanorods were captured as expected by quenching the high-pressure phase under ambient conditions at room temperature.i.e. the aspect ratio is ∼2.5, with a length of 85.1 ± 11.7 nm and a width of 33.6 ± 3.9 nm (the inset of tsh = 5.57 ± 0.45 nm (the inset of 31 The MnS shell shows a fringe spacing of 0.316 nm that matches the (002) plane of hexagonal MnS.32 The top-view HRTEM images indicated the smooth growth of MnS on MnSe and the explicit coaxial core–shell structure rather than the sulfur doped MnSe pattern  the pattern . Energy- pattern .in situ synchrotron angle dispersive X-ray diffraction (ADXRD) measurements (Pnma) of MnS and MnSe, respectively (2+ spin state from high-spin (S = 5/2) to low-spin (S = 1/2).33 Such lattice collapse driven by adjustable orbital/spin-state responses is rare. This unusual phenomenon should be further explored to accelerate the structural design of novel functional materials.33We performed high-pressure treatment of core/shell MnSe/MnS nanorods at a pressure up to 33.4 GPa and monitored the phase transition and formation of the new phase by urements . Before urements . The supurements . The twoectively  using Riectively  at 2.3 aPnma (a = 5.902 (1) Å, b = 3.900 (3) Å, and c = 6.500 (5) Å. Moreover, lattice parameters of B31-type MnS were estimated to be a = 5.656 (1) Å, b = 3.662 (3) Å, and c = 6.344 (5) Å, which are highly consistent with the previous results.34 The two sets of ring-type diffraction patterns in the ADXRD pattern correspond to the planes of B31-type MnS and MnSe, indicating the entrapment of the core–shell nanostructures R and (200)R planes (inset of R and MnS (200)R lattices from the selected areas indicted in R planes is calculated to be 2.55 Å (red lines), which is slightly (ca. 4.1%) greater than that of the MnS (200)R planes . Since the B31-type structure obtained during the RS-to-B31 phase transformation at high pressure is energetically more stable, the irreversible transformation in decompression is supported by the HRTEM and the corresponding FFT patterns. The atomic structure of the B31-type MnSe core is surrounded by the B31-type MnS shell suggesting that both the new high-pressure phase and core/shell geometry were captured after the diamond anvil cell was recovered to the ambient conditions at room temperature B and (2−11)B planes (inset of B is formed. B and MnSe (101)B lattices from the selected areas in B planes was calculated to be 3.82 Å (green lines), which is slightly (ca. 3.5%) less than that of the MnSe (101)B planes . Based on the aforementioned results, a structural model of the as-prepared core/shell MnS/MnSe nanorods with the B31 phase is schematically illustrated on the right side of TEM images provided additional evidence for the formation of coaxial core/shell MnSe/MnS nanorods with the high-pressure phase and preservation of their subsequent dual structure Fig. S4. In prininset of . Fig. 3dperature . The selinset of . As indiEg in terms of Kubelka–Munk transformations:A and hν are the edge–width parameter and the incident photon energy, respectively.35 We found that the band gaps of core/shell NCs decrease over the compression cycle  and density functional theory (DFT) calculations on the energy differences among WZ, RS and B31-phase MnSe within the pressure range up to 50.0 GPa  atoms integrally formed a MnS(Se)6 octahedron. The MnS(Se)6 octahedra are connected by four edges and two vertices for the RS structure, and the adjacent MnS6 octahedra in the B31 phase mutually shared the same surface. Thus, the MnS6 octahedron in the B31 phase is arranged much more compact than that in the RS phase, which was greatly improved by the creation of zigzag MnS4 planes inside the MnS6 octahedron along the b-axis direction. The rearrangements in the structural buckling profoundly facilitate the phase transformation to accommodate the increased external pressure. Upon decompression, the unique nanostructures and the inevitable thermodynamic influence should be responsible for the retention of the high-pressure new phase by aggravating the fluctuation of the subtle enthalpy differences under atmospheric pressure.Pressure-driven phase transitions offer us a pivotal protocol to fabricate new-phase core/shell nanoarchitectures. To better understand and interpret our experimental findings, we performed 50.0 GPa . The resd Fig. S8. No imag33 However, the transitions are unclear during decompression so far. Fig. S9et al.33 After fully releasing the pressure to ambient conditions, pure B31-type MnSe can be obtained.Before clarifying the effect of the MnS shell, we first characterize the crystal structure of pure MnSe NCs under high pressure. In bulk MnSe, the transition sequence during compression has been reported to be RS-to-high-pressure intermediate (HPI) phase-to-B31 up to about 47.4 GPa, where the HPI phase was assigned to a tetragonal distortion structure at an applied pressure of 22.8 GPa.40 On the other hand, the transitions were nucleated on the nanocrystal surface, where sliding and/or flattening of crystal planes occur and proceed inwards with increasing pressure.36 For heterostructures, the interface between different structures can act as an initiation site of the core to facilitate the occurrence of pressure-driven solid–solid phase transitions.23,41 The large lattice mismatch of the RS-MnS shell and the HPI-MnSe core shows that the absence of HPI in the MnSe core can reduce the interface strain. Therefore, the absence of HPI in the MnSe core may arise from the presence of abundant MnS phase interfaces as the dominant initiation sites, and the comprehensive effects enforced the WZ-to-RS-to-B31 phase transitions.We further investigated whether the thickness of the MnS shell would affect the aforementioned experimental results. Typically, MnS/MnSe NCs with thin shells (∼3.5 nm) were held between the opposed diamond anvils at room temperature Fig. S10. Besidesin situ ADXRD that identified the WZ-to-RS-to-B31 phase transition path for the core/shell MnSe/MnS nanorods. The generated new high-pressure phase B31-type core/shell NCs were captured as expected by quenching the high-pressure phase to ambient conditions at room temperature. The morphology of core/shell nanorods could be maintained after the high-pressure treatment. First-principles calculations indicate that B31-MnS and MnSe are thermodynamically stable under high pressure, and can survive under ambient conditions owing to the synergistic effect of subtle enthalpy differences in RS and B31 phases and high surface energy in nanomaterials. This study not only provides a fundamental understanding of pressure-driven phase transformations at the atomic scale but also sheds light on the rational design of new-phase heterostructured core/shell nanomaterials through a clean and fast stress-driven nanofabrication technique.In summary, we elucidate a new-phase heterostructured core/shell NC paradigm that is fed back from the pressure-induced B31 phase retention of core/shell MnSe/MnS nanostructures to ambient conditions at room temperature. Structural insights for this phenomenon were obtained using X. Y. and B. Z. designed the project and supervised the work. Y. W., H. L., M. W., K. W., Y. S., Z. L., Z. N., X. Y. and B. Z. performed experiments and analyzed data. S. L. and J. S. T. performed the calculations.There are no conflicts to declare.SC-012-D1SC00498K-s001


The benefits of postoperative chemotherapy in patients with estrogen receptor (ER)-positive breast cancer remain unclear. The use of tumor grade, Ki-67, or ER expression failed to provide an accurate prognosis of the risk of relapse after surgery in patients. This study aimed to evaluate whether a multigene assay Curebest™ 95GC Breast (95GC) can identify the risk of recurrence and provide more insights into the requirements for chemotherapy in patients.This single-arm retrospective multicenter joint study included patients with ER-positive, node-negative breast cancer who were treated at five facilities in Japan and had received endocrine therapy alone as adjuvant therapy. The primary lesion specimens obtained during surgery were analyzed using the 95GC breast cancer multigene assay. Based on the 95GC results, patients were classified into low-risk (95GC-L) and high-risk (95GC-H) groups.The 10-year relapse-free survival rates were 88.4 and 59.6% for the 95GC-L and 95GC-H groups, respectively. Histologic grade, Ki-67, and PAM50 exhibited a significant relationship with the 95GC results. The segregation into 95GC-L and 95GC-H groups within established clinical factors can identify subgroups of patients using histologic grade or PAM50 classification with good prognosis without receiving chemotherapy.Based on the results of our retrospective study, 95GC could be used to evaluate the long-term prognosis of ER-positive, node-negative breast cancer. Even though further prospective validation is necessary, the inclusion of 95GC in clinical practice could help to select optimal treatments for breast cancer patients and identify those who do not benefit from the addition of chemotherapy, thus avoiding unnecessary treatment.The online version contains supplementary material available at 10.1186/s12885-021-08778-5. Breast cancer is the most common cancer among women worldwide . The oveIn the past, the risk of breast cancer relapse was assessed by taking into account the patient’s age, tumor diameter, tumor grade, Ki-67, and ER expression. However, the risk of relapse and the need for chemotherapy are difficult to predict accurately based solely on clinicopathological factors.Recently, it has been possible to more accurately predict recurrence risk in breast cancer patients by supplementing information from clinicopathological factors used in previous breast cancer multigene assays, with some already incorporated in clinical practice. Oncotype DX®  is currently a widely used multigene classifier that can be used to stratify patients with node-negative ER-positive breast cancer into low-, intermediate-, and high-risk groups based on the expression of 21 genes –8. AnothCurebest™ 95GC Breast  is a breast cancer multigene assay that uses DNA microarray to analyze the expression of 95 genes , 13 and Thus far, the benefits of 95GC as a prognostic factor in patients with ER-positive, node-negative breast cancer have been evaluated only in retrospective studies conducted in a single facility or from public databases. Here, we aimed to assess the use of 95GC in patients from five Japanese facilities with ER-positive, node-negative breast cancer who received endocrine therapy alone as adjuvant therapy. To examine the relationships between 95GC and pathological factors associated with low reproducibility or inter-pathologist agreement, a repeat test was conducted with a central pathology review for Ki-67 and histologic grade and repeated analysis using array data for HER2.This study aimed to evaluate relapse-free survival between the 95GC-L and 95GC-H groups and the differences in the clinical features of each group. It also aimed to analyze the outcome of 95GC regarding individual clinical features, such as histologic grade and intrinsic subtypes.This non-interventional, non-invasive, and single-arm retrospective multicenter joint study included patients from five facilities in Japan . The surgery was performed between January 19, 1999, and October 22, 2010, with a median follow-up period of 90 months. We followed the STROBE guidelines , aromatase inhibitors (letrozole and exemestane), luteinizing hormone-releasing hormone agonist (goserelin and leuprolide), and a combination thereof  were also included. The primary lesion specimens were collected 30 min after surgery and stored at − 80 °C until analysis. Patients who received neoadjuvant therapy (prior to surgery) and adjuvant therapy other than endocrine therapy were excluded. In principle, only endocrine therapy is administered for T1/T2, lymph node-negative, and ER-positive breast cancer. Chemotherapy is recommended for patients with histologic grade 3, but it may not be administered at the patient’s request.Frozen specimens from patients were delivered on dry ice from the participating facilities to Sysmex Corporation and cryosectioned using Tissue-Tek® O.C.T. compound . Sections measuring 6-μm thick were prepared from the top and bottom edges of the specimen and stained using hematoxylin and eosin, and the tumor cell percentage was calculated. The remaining specimen was reserved for later use for microarray measurement.RNA was extracted and purified from the preserved specimens using the RNeasy® Lipid Tissue Mini Kit . Only specimens that yielded > 17 ng/μL RNA, determined using the NanoDrop™ 2000 , and with RIN value > 5.0, calculated using the Agilent 2100 Bioanalyzer , were considered valid for the study. Microarray measurements were performed using the GeneChip™ 3’IVT PLUS Reagent Kit (Thermo Fisher Scientific) and GeneChip™ Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific). All procedures were performed in accordance with the manufacturer’s protocol.P ≥ 30, spike-in labeling control lys_3_signal < phe_3_signal < dap_3_signal, and spike-in hybridization control BioB_3_signal < BioC_3_signal < BioD_3_signal < cre_3 signal. After normalizing the microarray data to analyze recurrence in patients using the RefRMA model, the 95GC algorithm was used to obtain the classification result of 95GC-L or 95GC-H, as described by Naoi et al. [The microarray data obtained were evaluated for quality using MAS5 normalization, and only the data that complied with the following conditions were considered: GAPDH3’/5′ ratio < 3.0, average background value in the range of 20–100, scaling factor ≤ 15, percent i et al. .Using the Kaplan–Meier method, the relapse-free survival curves of the 95GC-L and 95GC-H groups were plotted and evaluated using the log-rank test.The correlation between clinical factors and 95GC was evaluated using Fisher’s exact test. The hazard ratio for recurrence was evaluated for each factor based on the Cox proportional hazard model, and multivariate analysis was performed using factors with statistically significant hazard ratios in the univariate analysis. Each clinical factor was analyzed, excluding the missing data values. Multivariate analysis only included patients with available data on all the evaluated factors. The sample size was determined by calculating the number of cases required to verify the predicted 5-year recurrence-free survival rate of Curebest 95GC Breast .For patients with histologic grade 2 and those with the PAM50 luminal B subtype, relapse-free survival curves were evaluated for each 95GC risk group using the Kaplan–Meier method and log-rank test.All statistical tests were performed using two-tailed tests, with a significance level of 5%. We used the following applications: for Fisher’s exact test, R ver. 4.0.2 ; for the Cox proportional hazard model, Kaplan-Meier method, and log-rank test, MedCalc ver. 12.7.0.0 .A total of 102 patients met all the selection criteria. Samples for which 95GC assessment results could not be obtained owing to problems with RNA quality were excluded, and ultimately 75 patients were selected for analysis. We followed the method described in the study by Parker et al.  to deterThe relationships between the distribution of each clinical factor and the 95GC results in the analyzed patients are shown in Table p = 0.0017). The 5-year relapse-free survival rates of the analyzed patients were 97.8% for the 95GC-L group and 81.0% for the 95GC-H group. The 10-year relapse-free survival rates were 88.4% for the 95GC-L group and 59.6% for the 95GC-H group  between the 95GC-L and 95GC-H groups , the 10-year relapse-free survival rate for 95GC-L was 96.3%, which was significantly better than that for 95GC-H, and the relapse rate was extremely low. Therefore, chemotherapy can be considered omissible in patients with a histologic grade of 2 if they are 95GC-L.By performing a multivariate analysis of 95GC and clinicopathological factors utilized in previous studies to assess breast cancer recurrence risk, such as age, tumor diameter, tumor grade, Ki-67, and PAM50, we were able to confirm that 95GC is a significant independent prognostic factor that influences the recurrence risk. In our cohort we did not include patients that have received neoadjuvant therapy. Because neoadjuvant chemotherapy is often added in tumors ≥3 cm, the T2 cohort included only cases with a size close to T1 stage, and as a result, there was no difference in prognosis between T1 and T2 stages.The 95GC assay could provide extremely useful information for the selection of therapeutic strategies, such as adjuvant chemotherapy. The 95GC-L group had a better prognosis in response to adjuvant endocrine therapy than the 95GC-H group. Further prospective studies are needed to evaluate whether 95GC assays could be used to determine the necessity of chemotherapy in patients with ER-positive, node-negative breast cancer.The Oncotype DX®, a breast cancer multigene assay, analyzes 21 genes using reverse transcription polymerase chain reaction, and classifies patients by recurrence score (RS) (0–100) into low-, mid-, and high-RS groups. It is a useful prognostic factor in hormone receptor-positive, HER2-negative, node-negative breast cancer and a factor for predicting the effectiveness of adjuvant chemotherapy in the high-RS group , 6. NaoiIn everyday clinical practice, if a multigene assay is not available, Ki-67 is often used as a prognostic factor for ER-positive, HER2-negative breast cancer. In our study, Ki-67 measurement was performed through a central pathology review, but it was not a significant prognostic factor. It is difficult to establish a standardized method for evaluating Ki-67; therefore, it is difficult to accurately assess recurrence risk based on this factor. In the future, multigene assays for breast cancer should be incorporated more widely in everyday clinical practice.With regard to the usefulness of 95GC as a predictive factor for the effectiveness of chemotherapy, Tsunashima et al. examined 72 patients from their facility and 287 public database patients who underwent neoadjuvant chemotherapy. They reported a significantly greater tumor-shrinking effect in the 95GC-H group than in the 95GC-L group . Even thThe main limitation of this study was its small sample size. Additionally, the retrospective nature of the study resulted in a selection bias. Moreover, because of the archived samples, the quality of which was not controlled owing to the surgical practices used in the earlier days, 26.5% (27/102) of the samples had poor RNA quality. However, in recent times, the strict rules enforced for the handling of post-extraction specimens have helped to preserve RNA quality and, thus, guarantee a high rate of sample recuperation in everyday clinical practice. Finally, all study participants were Japanese, and the performance when targeting other races in other countries is unknown. However, a recently published study evaluated the recurrence stratification of patients from both Japan and the US, with comparable results between populations .Currently, 95GC has been available in Japan since 2013 for research purposes. Hence, further validation of the benefits of 95GC through a prospective study using a larger sample size is warranted to confirm the validity of incorporating 95GC in everyday clinical practice in Japan.This retrospective study suggests that 95GC is a long-term prognostic factor for ER-positive, node-negative breast cancer. To more optimally treat breast cancer patients, a system must be established to accurately assess the patient’s prognosis and determine whether the addition of chemotherapy will likely provide beneficial effects.Additional file 1. Strobe checklist.


Lipid management is the first line of treatment for decreasing the incidence of cardiovascular events in patients with coronary heart disease (CHD), and a variety of indicators are used to evaluate lipid management. This work analyses the differences in LDL-C and apoB for lipid management evaluation, as well as explores the feasibility of skin cholesterol as a marker that can be measured non-invasively for lipid management.n=53) and Group II (n=68) according to whether they had comorbid hyperlipidemia and/or diabetes mellitus. All patients were administered 10 mg/day of rosuvastatin and observed for 12 weeks. Lipid management was assessed on the basis of LDL-C and apoB, and linear correlation models were employed to assess the relationship between changes in these well accepted markers to that of changes in skin cholesterol.The prospective study enrolled 121 patients who had been diagnosed with acute coronary syndrome (ACS) at the department of emergency medicine of the First Affiliated Hospital of the USTC from May 2020 to January 2021, and the patients were grouped into Group I (P<0.05). LDL-C attainment rate is lower than apoB attainment rate with rosuvastatin therapy (P<0.05), which is mainly attributable to patients with low initial LDL-C. Skin cholesterol reduction correlated with LDL-C reduction.  and apoB reduction . Skin cholesterol reduction continued over all time points measured.Out of 121 patients with ACS, 53 patients (43.80 %) had combined hyperlipidemia and/or diabetes mellitus (Group I), while 68 patients (56.20 %) did not (Group II). Cardiovascular events occur at earlier ages in patients with CHD who are comorbid for hyperlipidemia and/or diabetes (Examination of changes in apoB levels give patients with low initial LDL-C more informative data on lipid management than LDL-C readings. In addition, non-invasive skin cholesterol measurements may have the potential to be used independently for lipid management evaluation. Over the past decades, China has achieved remarkable socio-economic development. However, in terms of health, it has shown a rapid increase in the morbidity and mortality of chronic diseases that are mainly part of atherosclerotic cardiovascular disease (ASCVD) . DyslipiIn 2005, Tzou’s team introduced skin cholesterol to assess the risk of cardiovascular events . Studies2) were excluded from the study.154 patients diagnosed with acute coronary syndrome (ACS) between January 2020 and April 2021 were included in the study. According to the exclusion criteria, 121 patients were finally enrolled : patients had comorbid diabetes and/or hyperlipidemia; and Group II (n=68): patients did not have diabetes or hyperlipidemia.Patients were divided into two groups based on the 2019 ESC lipid guidelines which recommend that patients with hyperlipidemia or diabetes have apoB evaluated due to false positive results with LDL-C readings . Group IAll patients received 10 mg/day of rosuvastatin as the starting lipid-lowering regimen. Patients with LDL-C <1.8 mmol/L and ≥50 % reduction at the 4th week of follow-up were considered to have achieved the LDL-C standard . ApoB atThe protocol was approved by Ethics Committee of University of Science and Technology of China (Registration No. 1804h08020291(2018-05-30)). Quantification of skin cholesterol was determined by specifically binding reagents to the skin, causing the cholesterol to fluoresce under excitation light  / Fisher’s exact test. Paired t-test was used for comparison of continuous variables within the group. Linear correlation analysis was performed between skin cholesterol reduction and LDL-C reduction, and skin cholesterol reduction with apoB reduction. A p-value <0.05 was rated as statistically significant.Repeated measure 1-way ANOVAs with Tukey post-tests were performed to compare data from different time points. For the categorical variables, the comparison was made using the chi-square test . There were 24 smokers in Group I and 22 in Group II, all of whom were male. Smokers accounted for 45.28 % and 39.29 % of male patients in each group, respectively (P<0.05). No statistical significance was found regarding gender composition nor history of hypertension . The apoB on admission was higher in Group I (1.01±0.48 g/L) than in the Group II (0.86±0.23 g/L) (P<0.05). There were not any differences between groups in high density lipoprotein cholesterol (HDL-C), LDL-C, number of low initial LDL-C cases, apolipoprotein A-1 (apoA-1), lipoprotein (a) (Lp (a)), nor initial skin cholesterol at admission (Table Patients in Group I had higher triglycerides (TG) (2.25±2.04 mmol/L ) than Group II (1.44±0.51 mmol/L) at admission (P<0.05). The 68 patients from Group II included 33 patients with apoB (+) and 35 patients with apoB (-), 20 patients with LDL-C (+) and 48 patients with LDL-C (-) ( P<0.05)  and 23 patients with apoB (-), 20 patients with LDL-C(+) and 33 patients with LDL-C (-) (P<0.05). The 12 patients from Group II included 1 patient with LDL-C (+)/apoB (+), 0 patient with LDL-C (+)/apoB (-), 6 patients with LDL-C (-)/apoB (+), 5 patients with LDL-C (-)/apoB (-) (P<0.05)  /apoB (+), 0 patients with LDL-C (+) /apoB (-), 6 patients with LDL-C (-) /apoB (+), and 3 patients with LDL-C (-)/apoB (-) (P<0.001) implying that skin cholesterol reduces in conjunction with LDL-C reduction   Fig. .Fig. 4LP=0.047, P=0.038), and portrayed a stable trend between the 8th week and 12th week  decreased over all time points measured  patients in Group I had a continual reduction in skin cholesterol over the first 8 weeks P=0.04, P=0.038With the progress of society, morbidity and mortality from CHD are increasing. Dyslipidemia is closely related to the development of CHD. Currently, guidelines recommend LDL-C as a therapeutic marker for the evaluation of lipid management and cardiovascular event risk status , 20. HowIn this study, patients with comorbid hyperlipidemia and/or diabetes accounted for 43.80 % of the patients recruited during the same period, and the mean age of onset was 56.43 years old in Group I, significantly lower than the average 62.26 years old in Group II. This suggested that patients with more risk factors suffered earlier CHD incidents than those with fewer risk factors, similar to the findings of recent studies .The assessment of lipid management outcomes using LDL-C and apoB showed significant differences between Group I and Group II. Further analysis revealed differences mainly in the results of the evaluation of patients with low initial LDL-C. Patients with low initial LDL-C on a 10 mg/day rosuvastatin regimen had trouble achieving the desired goal after 4 weeks of treatment and had difficulty meeting the LDL-C evaluation compliance requirements. However, those patients had absolute serum LDL-C values within the ideal range and were at low risk of recurrent cardiovascular events. This data suggests that it might be more feasible to use apoB to assess lipid management status in patients with initial low LDL-C.The study here within evaluated skin cholesterol as another marker. In a previous study, the team analyzed 342 study subjects  and found that skin cholesterol was associated with atherosclerotic disease , gender As research progresses, new LDL-C calculation equations and assays have been proposed , 7, 21. Evidence is provided within the current work which supports that apoB can be used for lipid management in patients with low initial LDL-C. Data is also provided depicting that skin cholesterol reduction is positively correlated with LDL-C reduction and apoB reduction. Therefore, skin cholesterol has potential value for lipid management evaluation in patients with CHD. Moreover, since the basic information of the patients in the study is similar to other studies, this work can be expanded to further develop non-invasive skin cholesterol testing in the future , 36.This study was limited by the follow-up time of each patient. This is due to guidelines requesting that lipid-lowering regimens need to be adjusted for patients who remain poorly controlled on lipid-lowering medications for 12 weeks. In addition, the study suffered from the shortcomings of a single center, small sample, and short duration.The current study analyzed the differences in the application of LDL-C and apoB and provided guidance for clinical work. In addition, the study identified the potential value of skin cholesterol for lipid management in patients with ACS, providing a reference for the selection of markers for lipid management. Moreover, use of skin cholesterol as a marker could enhance lipid management in patients with ACS since the method of detection is non-invasive.